Regulatory RNAs in immunosenescence

Abstract Background Immunosenescence is a multifactorial stress response to different intrinsic and extrinsic insults that cause immune deterioration and is accompanied by genomic or epigenomic perturbations. It is now widely recognized that genes and proteins contributing in the process of immunosenescence are regulated by various noncoding (nc) RNAs, including microRNAs (miRNAs), long ncRNAs, and circular RNAs. Aims This review article aimed to evaluate the regulatore RNAs roles in the process of immunosenescence. Methods We analyzed publications that were focusing on the different roles of regulatory RNAs on the several aspects of immunosenescence. Results In the immunosenescence setting, ncRNAs have been found to play regulatory roles at both transcriptional and post‐transcriptional levels. These factors cooperate to regulate the initiation of gene expression programs and sustaining the senescence phenotype and proinflammatory responses. Conclusion Immunosenescence is a complex process with pivotal alterations in immune function occurring with age. The extensive network that drive immunosenescence‐related features are are mainly directed by a variety of regulatory RNAs such as miRNAs, lncRNAs, and circRNAs. Latest findings about regulation of senescence by ncRNAs in the innate and adaptive immune cells as well as their role in the immunosenescence pathways, provide a better understanding of regulatory RNAs function in the process of immunosenescence.


| IMMUNOSENESCENCE 1.| Definition and criteria
Immunosenescence refers to the alterations of the innate and adaptive immune components mainly caused by normal age advancement. 1 During the early 1960s, Walford found that immune responses decrease with aging and contribute to increased susceptibility to infection, chronic age-related and autoimmune diseases as well as malignancies, which was later published in his book known as "The immunologic theory of aging." 2 The immunosenescence process is not a random phenomenon, but it is appeared to be conserved during evolution and is strictly controlled by genetic and epigenetic factors. 3This process is associated with organ alteration and modified phenotypes and functions of various immune cells.The main features of immunosenescence are cell growth arrest, apoptosis resistance, senescence associated secretory phenotype (SASP), decreased telomere length and telomerase activity, elevated SA-βgalactosidase activity, increased expression levels of P16 and P21, decreased CD27/CD28 expression, increased CD57/Killer cell lectin-like receptor subfamily G (KLRG-1) expression, elevated glycolysis and reactive oxygen species production and reduced mitochondrial metabolic pathway. 42 | Impact on the immune system An increasing number of studies have suggested that immunosenescence is associated with chronic low-grade inflammation which is described as inflammaging.5 Inflammaging is characterized by upregulated blood and tissues inflammatory markers as a result of constant antigenic challenge and considered as a significant risk factor of mortality and morbidity in aged individuals.6,7 Cellular senescence, mitochondrial dysfunction, defective autophagy, inflammasome activation, induction of the DNA damage response, alterations in the microbiome composition, and chronic infections such as human cytomegalovirus (CMV) are responsible for inflammaging related to immunosenescence.8,9 Whereas adaptive immune responses are more affected by immunosenescence, innate immune responses also change during this process.Certainly, senescent innate immune cells exhibit decline in antigen processing and presentation capacity, a reduced ability to respond to new antigens (Ags), reduction and delay in type I IFN production, reduced neutrophils phagocytosis and oxidative burst, decreased macrophage chemotaxis and phagocytosis, increased cytokine production and altered Toll-like receptors (TLRs) expression.4,10,11 A number of studies showed increased frequency of nonclassical CD14 + CD16 + monocytes with reduced surface expressions of HLA-DR and CX(3)CR1 in the elderly.][13][14] In addition, decreased IL-6 and increased IL-10 production level were demonstrated in the macrophages of aged mice, following stimulation with TLR ligands.Also, it was showed that aged macrophages have reduced number of CD14 + TLR4 + expressing cells as well as decreased levels of IL-6, IL-1β, and IL-12.15 Interestingly, van Beek et al. found that aging can induce the accumulation of alternatively activated (M2-like) macrophages, with proinflammatory phenotype in tissues, which express senescence markers.Therefore, aging in macrophages affects different functions such as TLR signaling, phagocytosis, and wound repair.Despite the lack of significant differences in the number of myeloid DCs during aging, several studies show dramatic age-related reduction in the number and function of plasmacytoid DCs.[16][17][18] In response to foreign Ags, DCs from aged individuals displayed significantly reduced ability to Ags uptake.20][21] Aging also affects the phagocytic and chemotactic abilities as well as recruitment of neutrophils, which are important in early defense against invading pathogens (40, 44).Neutrophils of the elderly subjects display decreased neutrophil extracellular traps (NET) that are able to bind and trap extracellular pathogens to defend against infectious agents.22,23 Therefore, age-related reduction in NETosis can lead to delayed wound healing and increased susceptibility to invasive pathogens.The dysregulated responses in the adaptive immune cells during immunosenescence, include thymic involution, decreased frequencies of naïve T and B cells, accumulation of memory T cells, reduced expression of CD40 ligand and decreased antigen-specific antibody (Ab) production.24 Previous data reported limited diversity in B cell repertoire and also poor Ab response after vaccination in elderly individuals.[25][26][27] Also, human peripheral B cell frequency and numbers significantly reduce production in the bone marrow of aged subjects, while B lymphopoiesis is active throughout life.[28][29][30] Recent studies have elucidated profound and complex changes in aged T cells, including epigenetic and metabolic modifications, affecting different subsets such as naïve, memory, and effector T cells.[34][35] Additionally, it has been noted that aging-related T cell may show defects in cytokine production.36,37 Schmitt et al. reported the higher Th17 to Treg ratio during aging which is related to increased rate of autoimmune disease in the elderly.In particular, age-dependent increase in the ratio of Th17 cells to Treg cells may induce proinflammatory cells and contribute to autoimmune diseases.38 Recently, it has been shown aged PD-1 + /CD153 + CD4 + memory T cells, which are characterized by defective proliferation and cytokine production.39 Also aging can lead to decreased number of naïve CD8 + T cells and reduced diversity of the TCR repertoire during CMV infection.40

| Impact on proteostasis
Proteostasis or protein homeostasis is the process that regulates the biogenesis, folding, trafficking, and degradation of proteins present within and outside the cell. 41isruption of proteostasis leads to the accumulation of protein aggregates which are part of the process of development of many age-related diseases, prominently including Alzheimer's disease, cancer, and diabetes. 42ifferent quality control systems such as molecular chaperones, ubiquitin proteasome system, and autophagylysosomal system are involved in the regulation of proteostasis. 43These proteolytic systems decline during aging, indicating that proteostasis is a major hallmark of aging and cellular senescence. 44It is already known that chronic ER stress, insufficient unfolded protein response (UPR) and excessive apoptosis contribute to a deregulated proteostasis pathway in the cells. 45A recent study reported increased transcriptional activity of the stress-induced HSPA1A/B and HSPA6 genes and p62 protein content in the peripheral blood mononuclear cells (PBMC) of mild to moderate patients with Parkinson's Disease (PD).As p62 plays important roles in both autophagy and apoptosis pathways, the higher level of p62 in patients with PD may enhance the occurrence of spontaneous apoptosis in PBMCs. 46

| SASP
SASP is a phenotype related to senescent immune and nonimmune cells, characterized by the secretion of high levels of inflammatory cytokines, interleukins (IL), immune modulators, growth factors, angiogenic factors, microRNAs (miRs/MiRNAs) and proteases. 47SASP can re-enforce immunosenescence in immune cells and neighboring normal cells, in autocrine and paracrine manners, respectively. 48[51]

| NONCODING (nc) RNAS AND IMMUNOSENESCENCE
Given that immunosenescence influence the function and phenotype of immune cells that lead to perturbation of immune system responsiveness, understanding the critical regulators of immunosenescence may provide insight into the underlying mechanisms of immune-related diseases of aging.The ncRNAs are functional RNA molecule that are not translated into a protein. 52hey are one of the main components of epigenetic mechanisms during genes expression and they are participating in transcription, splicing, translation, modification and stability of other RNAs, regulation of chromatin structure as well as cellular proliferation, apoptosis and differentiation. 53Different types of ncRNAs are divided to housekeeping (rRNAs and tRNAs) and regulatory RNAs.Regulatory ncRNAs contain long ncRNAs (lncRNA), enhancer RNAs (eRNA), circular RNAs (cir-cRNA), and small ncRNAs such as miRNA, small nucleolar RNA, small nuclear RNA and PIWI-interacting RNA. 546][57] These RNAs by modulating major senescence-associated pathways such as p53/p21, pRB/p16 and SASP are contributed in aging and aging-related diseases. 55,58In the following sections, we discuss the MiRNAs, lncRNAs, and circRNAs that affect the SASP phenotype and SASP gene expression and provide current evidence that their dysregulation contributes to the process of immunosenescence.
MiRNAs are a family of small, single-stranded, nc RNA molecules containing 21−23 nucleotides that regulate gene expression at the post-transcriptional level. 59MiRNAs are synthesized in a multistep process by RNAi machinery, including RNA Polymerase II, DiGeorge syndrome critical region 8/Drosha heterodimer, Exportin 5 (XPO5)/RanGTP and RNAse Dicer, which participate in the formation of long pri-miRs containing stem loop structures, nuclear cropping of the long pri-miR into smaller 60-100 bp pre-miRs, nuclear export of pre-miRs and their cytoplasmic maturation into 23 bp mature miRNAs, respectively. 60MiRNAs are usually complementary to 3′ untranslated region of target mRNA, promoting degradation of mRNA by RNA-induced silencing complex. 61The miRNAs play important regulatory roles in the development of hematopoietic lineage, 62 proliferation of neutrophils and monocytes, 63 secretion of inflammatory cytokines, 64 and proficiency of innate and adaptive immune responses. 65Hence, according to the regulatory roles of miRNAs, they probably contribute in immunosenescence regulation.

| MiRNA regulated thymic involution
While the precise mechanism of thymic involution remains unclear, the involvement of different miRNAs in thymic aging involution have been reported. 66It has been indicated that miR-181a-5p is downregulated in thymic epithelial cells (TECs) of aged mice, which is associated with age-related thymic involution by directly targeting of transforming growth factor β receptor 1 and Smad family member 3. 67 Moreover, it has been found that the expression levels of some MiRNAs such as miR-25, miR-7f, and miR-134 were decreased in the thymus of 70-year-old men as compared with <10-month-old newborns.These data suggested that these MiRNAs, through modulating of WNT and FoxN1 signaling pathways, participate in age-related thymic involution. 68A previous study using 1892 unique probes for comparing altered MiRNA expression profiles between young and aged mice TECs, identified that miR-125a-5p, was increased in aged thymi. 69Another study also indicated that miR-29a cluster deficiency leads to increased sensitivity of the TECs to poly (I:C) and premature thymic involution. 70n addition, higher expression level of miR-2137 was detected in mice TECs that gradually increased during the process of thymus aging. 71

| MiRNA regulated proteostasis
The role of MiRNAs in the proteostasis regulation such as translation, folding, and degradation has been reported recently. 72A previous study found that miR-425 interacts with heat shock protein B8 (HSPB8) and promotes tau phosphorylation in HEK293/tau cells which is related to senescence-associated Alzheimer's disease. 73Furthermore, organelle stress responses like ER stress contribute to the maintenance of proteostasis.Aging and cellular senescence are associated with a prolonged UPR and ER stress, which is regulated by several MiRNAs.A recent study found that overexpression of miR-23a∼27a∼24-2 cluster was related to ER stress-mediated apoptosis pathway, including C/EBP homologous protein (CHOP/DDIT3/GADD153) and TRIB3, an Akt inhibitor and ATF4 molecules. 74It has been shown that higher expression level of miR-204 attenuated the induction of several ER-responsive genes such as GRP78, GRP94, and CHOP that was associated with phenotypes of senescent cells. 75The regulatory role of MiRNAs in the control of autophagy during aging has been reported in several studies.In Caenorhabditis elegans, miR-83/miR-29 were associated with agerelated decrease in autophagy across different tissues. 76n addition, another study demonstrated that miR-34-5p prevents mitophagy via targeting Pink1 and leads to the age-related downregulation of mitophagy in aged mice. 75

| MiRNA regulated SASP production
As mentioned above, SASP is characterized by increased secretion of several cytokines, growth factors and matrix metalloproteases. 47Recently, different types of MiRNAs have been shown to regulate the secretion of SASP in senescent immune cells. 77MiR-187 was found to be more highly abundant in senescent human monocytes which inhibited the production of major SASP factors, such as TNF-α and IL6. 78Senescent human PBMCs showed increased secretion of miR-21 in response to lipopolysaccharide (LPS), leading to reduced NF-κB activity and increased IL-10 production. 79The expression level of miR-125b was decreased in LPS-stimulated RAW 264.7 macrophages.Further analysis revealed that miR-125b reduces the level of TNF-α and consequently LPSdependent decrease in miR-125b level may be involved in the LPS-induced increase in TNF-α and SASP. 80oreover, LPS triggered miR-147 expression in mouse macrophages through the TLR4-NF-κB signaling pathway.It has been reported that miR-147 overexpression inhibits the excessive production of TNF-α and IL6 inflammatory cytokines in macrophages. 81

| MiRNA regulated inflammaging
Several MiRNAs play an important role in modulating inflammaging during cellular senescence. 82,83][86][87] Previous studies confirmed that miR-21−5p can target programmed cell death 4 which contributes in the activation of NF-κB and IL-6 production, as well as inhibition of antiinflammatory cytokine IL-10. 88,89A recent study indicated that inhibition of miR-570-3p in human primary small airway epithelial cells leads to the partial recovery of cellular senescence, with improved cellular growth and reduced expression of SASP inflammatory proteins, including MMP-2/9, IL-1β, CXCL8, and IL-6. 90

| MiRNA regulated macrophage senescence
Chronic low-grade inflammatory conditions during immunosenescence is due to overexpression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)induced cytokines, including IL-6, IL-1β, and TNF-α, which are primarily secreted by macrophages. 3In a previous study, higher expression levels of miR-146a in un-stimulated macrophages of aged mice as compared with that of young mice has been found. 91Furthermore, miR-146a in macrophages of aged mice regulates IL-6 and IL-1β secretion by targeting the IL-1 receptor-associated kinase 1 (IRAK1) level. 92It has been indicated that miR-150 is upregulated in aged murine peritoneal, splenic and BM-derived macrophages and induces differentiation of aged macrophages toward disease-prone phenotype. 93A recent mice study clarified that the expression levels of miR-146b in thioglycolate-elicited macrophages were reduced during aging and that may contribute to age-associated inflammation and cellular dysfunction. 94

| MiRNA regulated T cell development, differentiation and plasticity
5][96][97] MiR-181a plays a major role in both positive and negative selection of double positive cells during thymic development of immature T cells.][100] MiRNA expression profiling in mouse showed that miR-150 inhibits the early stages of T cell development. 101Furthermore, it has been shown that miR-150 targets transcription factors c-Myb and NOTCH3, resulting in decreased T cell proliferation and survival. 102Both TCR-MHC antigen complex and costimulatory signals induce T cells activation.Cytokines such as IL-2, which is an essential factor for T cell activation, are shown to be under transcriptional control of miR-155, miR-181c, miR-9 and miR-31. 103MiR-9 has been shown to directly target B-lymphocyte-induced maturation protein 1 and BCL6 and therefore modulates T cells activation. 104iR-146a is induced upon TCR activation, negatively regulates the expression levels of TNF receptor-associated factor 6 and IL-1 IRAK1 and therefore increases T cells activation. 105In addition, several miRNAs are known to regulate different peripheral T cell subsets. 67It has been shown that miR-132, miR-200, and miR-212a regulate IL-12 production which required for differentiation of Th1 cells. 106Overexpression of miR-125b was shown to promote Treg cells differentiation and suppress Th17 cells differentiation. 107Two members of Let-7 family, including let-7g-5p and let-7d-3p regulate Th17 differentiation and IL-17 expression through targeting STAT3 and AKT1/ mTOR signaling pathways, respectively. 108,109It has been reported that miR-17-92 cluster decreases Th1 cells differentiation via reducing T-bet and IFN-γ expression whereas promotes Foxp3+ Treg differentiation. 110

| MiRNA regulated T cell senescence
During immunosenescence, T cells downregulate the expression of surface markers such as CD27, CD28, CCR7 and CD45RO, while they upregulate the KLRG-1, CD57, PD-1, and CD153. 1113][114][115][116][117] A previous study found that the miR-17~92 cluster is downregulated in aged CD8 + CD28 + T cells compared with young CD8 + CD28 + T cells, which is correlated with replicative senescence in aged T cells by increasing the level of p21 protein. 113Furthermore, decreased expression level of miR-92a in naive CD8 + T cells was found to correlate with aging. 115Another study reported downregulation of miR-181a in aged CD4 + T cells that was related to the threshold of TCR signaling and impaired TCR sensitivity. 114In addition, a recent study showed increased expressions of miR-9-5p, miR-34a-5p, and miR-23a~24-2 cluster in naive CD28 + T cells upon IL-15 stimulation, which correlated with loss of CD28 in T cells and specific characteristics of T cell aging. 118Certainly, IL-15 is a homeostatic cytokine that is capable of downregulating CD28 expression level by inducing TNF-α production and alteration of CD28 promoter activity. 119In addition, IL-15 can induce MiRNAs interacting with the 3' UTR of CD28 mRNA and results in loss of CD28. 118

| LncRNA
LncRNA are a class of RNA molecules containing more than 200 nucleotides that are not translated into protein.
1][122][123] LncRNAs are expressed during cell differentiation and development which makes them capable of regulating the cell cycle, DNA replication timing and chromosome stability, genetic imprinting, stem cell reprogramming, chromatin remodeling, gene transcription, mRNA stability, translation, and protein stability. 124Therefore, lncRNAs may affect several cellular processes, including cellular proliferation, differentiation, senescence, quiescence, and response to stress and immune agents. 125

| LncRNAs regulated proteostasis
Different types of lncRNAs influence protein trafficking by translocating transcription factors to the nucleus and/ or to certain DNA regions.In the previous study, higher levels of lncRNA PANDA was reported upon DNA damage during cellular senescence.PANDA binds to the NF-YA transcription factor, interfering with its transcription and lowering the expression of apoptotic genes.These data suggest that PANDA might be involved in DNA damage-induced senescence through NF-YA and p53 Di. 126,127 Another study indicated that reduced interaction of lncRNA 7SL with TP53 mRNA in human cervical carcinoma HeLa cells and human pancreatic adenocarcinoma MiaPaCa cells leads to promoted p53 translation and in turn increased cell cycle arrest and cellular senescence. 128oreover, several lncRNAs interact with proteins, RNA, and DNA to regulate protein assembly.It has been reported that the lncRNA TERC is crucial for telomere formation and telomere length maintenance and thus the inhibition of premature senescence and aging. 129,130Indeed, TERC serves as a template for telomerase to synthesis the telomeric repeats. 131

| LncRNA regulated SASP production
Higher expression levels of long intergenic nc RNA cyclooxygenase 2 (lincRNA-Cox2) and the TLRs (TLR2/ TLR1) agonist, Pam3CSK4 were reported in bone marrowderived macrophages after treatment with LPS via stimulating NF-κB and the TLR adaptor myeloid differentiation primary response 88 (MYD88).However, lincRNA-Cox2 by interacting with the RNA-binding proteins heterogeneous nuclear ribonucleoproteins (hnRNP A/B and hnRNP A2/ B1), suppresses immune gene transcription, which can induce IL-6 expression via TLRs signaling. 132These findings suggest that lincRNA-COX2 may affect aging and age-related diseases by playing dual roles in activation and inhibition of immune response genes in macrophages.A recent study in THP1 macrophages showed that the interaction of lncRNA-THRIL (TNF-α/hnRNPL-related immunoregulatory lncRNA) with the RNA-binding protein hnRNP-L, triggers TNF transcription through binding to the TNF gene promoter. 133

| LncRNA regulated inflammaging
8][139][140] It has been reported that lncRNA Lethe binds to the NF-κB subunit RelA and inhibits the production of inflammatory proteins. 141Moreover, Lnc-IL7R was found as an inducer of LPS-associated inflammatory response.Indeed, a previous study indicated that depletion of Lnc-IL7R led to the reduced levels of inflammatory mediators, including E-selectin, VCAM-1, IL-6, and IL-8. 142Therefore, lncRNAs may play a role in inflammaging by regulating the NF-κBrelated proinflammatory cytokines secretion by senescent cells.

| LncRNA regulated T cell development, differentiation and plasticity
A recent study showed that linc-MAF-4 plays a key role in Th1 cells differentiation and that knockdown of linc-MAF-4 in naive CD4 + T cells results in expression of the Th2specific genes like MAF, GATA3, and IL-4. 143Moreover, LincR-Ccr2-5' AS, induces the expression of Th2-specific gene downstream of GATA3 signaling, Including Ccr1, Ccr3, Ccr2, and Ccr5. 144A previous investigation reported that overexpression of IFNG-AS1 in CD4 + T cells led to elevated inflammatory cytokines secretion, reduced levels of antiinflammatory cytokines and increased differentiation to Th1/ Th17 cells through targeting miR-125a and upregulating IL-23R. 145The lncRNA Th2-LCR found to regulate the production of cytokines associated to differentiation of Th2 cells by translocating to IL-4, IL-5 and IL-13 coding genes, and its knockdown reduced H3K4 methylation of the IL-4 and IL-13 promoters. 146,147Several lncRNAs have been reported to modulate the T cells function.For instance, lncRNA NRON regulates the expression of nuclear factor of activated T cells (NFAT1) that is required for IL-2 production as well as naive T cell proliferation and survival. 148

| CircRNAs
CircRNAs are single-stranded, covalently closed RNAs formed by cellular spliceosomal machinery which joins a downstream splice donor to an upstream splice acceptor. 149ircRNAs share a set of general functions, including proteins and peptides translation, cell metabolism, protein translocation and protein-protein interactions. 150CircRNAs impede cellular proliferation by binding to the cell cycle proteins cyclin-dependent kinase 2, cyclin-dependent kinase inhibitor 1 (p21), and mammalian forkhead transcription factors. 151Moreover, circRNAs affect cellular survival through programmed cell death involving apoptosis. 152hese data suggest that circRNA may participate in the regulation of aging and age-related diseases.

| CircRNA regulated T cells senescence
A previous study employed high-throughput circRNA microarrays to investigate profiles of differentially expressed circRNAs in CD28 − CD8 + T cells in the elderly and adult subjects. 153Their results showed that circ-100783 may regulate phosphoprotein-related signal transduction in CD28-dependent CD8 + T cells which is related to loss of CD28 in CD8 + T cells during aging. 153

| CONCLUSION
Immunosenescence is a complex process with pivotal alterations in immune function occurring with age and is associated with poor response to vaccinations and increased sensitivity to diseases.The immune dysfunction that characterize the immunosenescence process are governed by specific changes in the pools of expressed genes and proteins.Therefore, there is growing interest in understanding the extensive network of mechanisms that drive immunosenescence-related features.Immunosenescence is a complex process with pivotal alterations in immune function occurring with age.These processes are mainly directed by a variety of regulatory RNAs such as miRNAs, lncRNAs, and circRNAs.Certainly, regulatory RNAs are involved in the aging of immune system by regulating immune cells differentiation, activation and function as well as secretion of inflammatory cytokines/chemokine and effectiveness of immune system response.Although emerging evidence has been accepted the regulatory RNAs roles in regulation of immunosenescence; but, regulatory RNAs characteristics in detail, role of each RNAs, and their exact targets in the immunosenescence process are not clearly investigated.Therefore, studies discovering this complex process or determining methods for modulation of regulatory RNAs expression during the immunosenescence may donate possible therapeutic approaches in prevention and/or treatment of ageassociated life-threatening diseases.